Mass spectrometry imaging. What does MS imaging offer?
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1 BMG 744 Mass spectrometry imaging Stephen Barnes, PhD With sincere acknowledgments to David Stella, PhD and Kyle A. Floyd, MS, former students in the Barnes Laboratory ( ) and Kevin Schey, PhD, Vanderbilt University What does MS imaging offer? Can provide information that grind and find cannot What is in the imaged section? Where is it? How much of it is there? Is it modified? As we ll see, much as the laser is targeted at the frozen section, it is an untargeted assay 1
2 Imaging is widely used in research Light microscopy 2
3 Confocal Microscopy Electron microscopy 3
4 Mass spectrometry imaging A chemical image Principle of MALDI imaging (Caprioli, 1997) 4
5 A simple example of MS imaging Coomasie Blue image Image based on the detection of the ion for Coomasie Blue Interpolated image reconstruction (Caprioli, 1997) Modern applications of MS imaging 5
6 How to get analytes off a tissue section Several types of ionization sources are used: SIMS Secondary Ion MS DESI Desorption electrospray ionization MALDI MALDESI LAESI LaserAblation Electrospray Ionization SIMS 6
7 DESI MALDI 7
8 MALDESI (KK Murray) LAESI 8
9 Profiling versus imaging Profiling: Limited, it directed d information Rapid analysis high throughput Clinical applications and biomarker discovery Imaging: Extensive, high resolution Time consuming, laborious Useful for investigative research MALDI matrices 9
10 Matrix solvent How to spot the matrix 10
11 AIMS 2012 Vanderbilt U AIMS 2012 Vanderbilt U 11
12 Cataract disease in an Amazonian Cataract Disease and Public Health: WHO states that age related cataract is responsible for 48% (~22 million people affected) of blindness in the world. Today people are living i longer, putting more elderly at risk for cataract disease. Currently surgical removal is the only treatment, for which many countries have inadequate resources. In 2004 U.S. alone, cataract related medical expenses (i.e., surgical lens replacement) were estimated at $6.8 billion dollars, or ~42% of total vision related costs. (Rein et al. 2006). Most common surgery in USA (431,000) Map from WHO: Picture from WHO: 12
13 Lens cataract incidence with aging Keenan et al., Br J Ophthalmol 2007;91: Lens changes occur in pets, too Cloudiness is a natural event that occurs with aging and precedes cataract formation. Due to packing of the lens fiber cells. 13
14 Anatomy of the eye Issues that relate to the lens 14
15 The rat model we use MALDI TOF MS profiling of nuclear region of SD rat lens Intensity % % Intensity Nuclear day Increased crystallin fragments Nuclear day Mass (m/z) 1.5E+4 2.7E+4 15
16 Acoustic spotting onto frozen lens section Pickett et al Spotting to the frozen rat lens Aerni et al Our Deposition Method: Overlay squares of 400 μm, for a final array resolution of 200 μm. 40 cycles of 1 drop per spot per cycle; 170 pl total volume per spot. Matrix: 50% Acetonitrile, 45% Water, 5% Formic Acid, 15mg/mL Sinapinic Acid, with/without 0.1% Triton X. 16
17 Lens Cataract: imaging reveals a geographic distribution of protein forms What are the proteins that imaging is detecting? Stella et al., 2010 Aqueous extract of the lens Stella et al.,
18 Water insoluble/urea soluble Stella et al., 2010 Top down identification of m/z= (5+ charge state) Stella et al.,
19 Top down identification of m/z= Isolate precursor ion ( Da) and fragment using collision induced dissociation Fragmentation spectrum of m/z = m/z Stella et al., 2010 Top down ID of (M+H + )= 6, Da A-crystallin MDVTIQHPWFKRALGPFYPSRLFDQFFGEGLF EYDLLPFLSSTISPYYRQSLFRTVLDSGISEVSPYYRQSL RSDRDKFVIFLDVKHFSPEDLTVKVLEDFVEI HGKHNERQDDHGYISREFHRRYRLPSNVDQSA LSCSLSADGMLTFSGPKVQSGLDAGHSERAIP VSREEKPSSAPSS A crystallin truncation (1 53): 6, Da (14 ppm) Fragmentation spectrum of m/z = m/z Stella et al.,
20 Top down ID of (M+H + )= 6, Da A-crystallin MDVTIQHPWFKRALGPFYPSRLFDQFFGEGLF EYDLLPFLSSTISPYYRQSLFRTVLDSGISEV RSDRDKFVIFLDVKHFSPEDLTVKVLEDFVEI HGKHNERQDDHGYISREFHRRYRLPSNVDQSA LSCSLSADGMLTFSGPKVQSGLDAGHSERAIP VSREEKPSSAPSS m/z Stella et al., 2010 Top Down Protein Assignment Full length Stella et al. (2010) Invest Opth Vis Sci:
21 Structure of A crystallin Distribution of truncated A crystallins Stella et al.,
22 Distribution of truncated A crystallins Stella et al., 2010 Clark et al., ARVO,
23 Optimization of spotting methods Plate Mounted, Vertical EtOH wash Plate Mounted, Horizontal EtOH wash with Blotting Carbon Fiber Tape Mounted and Lyophilized Slide adapted and modified from Kyle Floyd Matrix-spotted Tissue Sections 1-53 αa-truncation αa-truncation Full-Length αa-crystallin Overlaid Images: Red: 1-53 αa-truncation Blue: αa-truncation Yellow: Full-length αa-crystallin Normalized Average Spectrum Figure1: Tissue sections and selected images for adult female ICR/f rat with full cataracts. These five sections were taken from the same lens, with four sections taken between each imaged section, and saved for future proteomic analysis. 23
24 Small molecule imaging Imaging of lipids in tissue Fresh frozen tissue Frozen tissue sectioning Cut and transfer micron section to conductive glass slide DHB (MALDI matrix) is transferred by sublimation. This produces a very uniform coating (as shown in B) and high resolution images 24
25 Differential lipid distribution in the retina MALDI QIT TOF Hayasaka et al m sections sprayed with matrix (in MeOH) using an airbrush Using a higher resolution approach The same Japanese group used an evaporation method to apply the matrix see next slide 25
26 Sublimation method for applying MALDI matrix to the tissue slice MALDI plate is upside down ice is placed inside the glass finger to condense the DHB A cold trap (not shown) is needed after the heated chamber to protect the vacuum pump Sand bath to heat the DHB PC species distribution in the brain 26
27 Ceramides and ischemia by IMS The ion was determined to be Cer 18:0/18:1 ( H 2 O) Greving et al
28 Spectrum from a single cell Greving et al Extreme sensitivity Greving et al
29 Mass Spectrometry Imaging Workshop April 20 24, 2013 Vanderbilt University aims2013 Registration: $750 before Mar 15 $950 after Mar 15, up to Mar 31 29
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